Project Summary The central nervous system regulates gonadotropin secretion through neurosecretion of gonadotropin- releasing hormone (GnRH). GnRH is synthesized in preoptic and periventricular neurons, transported via intertwined processes that distribute as axonal-vascular terminals in the median eminence and released into the hypothalamic-hypophysial portal vasculature. GnRH binds receptors on gonadotropes to stimulate secretion and synthesis of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Neurosecretion of GnRH is pulsatile and is obligatory for sustaining normal gonadotropin secretion and synthesis. Kisspeptin (KISS1) neurons are a major GnRH afferent network that activates GnRH neurons during puberty, maintains GnRH release during adulthood, and transduces negative and positive feedback actions of gonadal steroids. In our studies we will test the hypothesis that KISS1 neurons, specifically those in the arcuate nucleus (ARC) that co-express the peptides neurokinin B (NKB) and prodynorphin (PDyn) (KNDy neurons), function as a GnRH pulse generator (Aim 1), with NKB mediating synchronized activation of KNDy cells (Aim 2) and dynorphin terminating each pulse discharge (Aim 3). These studies are significant because they will provide information on the cellular circuitries that govern GnRH pulse generation, and thus will shed new light on the pathophysiologic mechanisms underlying neuroendocrine-based fertility disorders, such as absent, delayed, or precocious puberty, functional hypothalamic amenorrhea, or polycystic ovary syndrome, and in the longer term, point to new potential targets for intervention in these reproductive impairments. The proposed studies will use powerful genetic tools to dissect and characterize the functional components of the kisspeptin-GnRH pulse generating system. To define the key elements of the pulse generator, we will utilize innovative genetically modified mouse models, each enabling an analysis of the consequences of kisspeptin neuron-specific gene deletion. We will first study the role of the KNDy neuronal population using a Kiss1fl/fl mouse recently created in our laboratory. This mouse will be bred to a prodynorphin Cre (Pdyn-IRES- Cre21,2) mouse, hereafter Pdyn-Cre, to eliminate Kiss1 in KNDy neurons. We have also developed a novel mouse bearing a doxycycline-inducible kisspeptin-Cre allele (iKiss-Cre mouse) that will enable temporal control over the selective deletion of genes from Kiss1 neurons. Two other lines, a NKBfl/fl mouse (developed in our laboratory) and an Oprk1fl/fl mouse (Jackson labs) will be crossed to Kiss1-Cre or iKiss-Cre mice to generate mice that will allow us to analyze the effects of kisspeptin neuron-specific deletion of NKB and Oprk1 on GnRH- triggered LH pulses. We will also use the iKiss-Cre mouse to delete and study the roles of steroid receptors in kisspeptin neurons in the adult. This temporally controlled gene deletion will eliminate a longstanding technical limitation of conventional steroid receptor knockout models in which steroid regulation of the axis is confounded by steroid developmental and organizational effects in the axis.